The great expectance aroused in the past few years for the so called cellular therapy and gene therapy is limited by current methods for the cultivation of specific cell types in vitro.
New methods for the maintenance, survival, proliferation and differentiation of the different cell populations are needed.
Generally, methods for cell culture make use of culture media supplemented with growth factors and cytokines which exert specific biological effects on cells. The cell culture medium may be any cell culture medium which adequately addresses the nutritional needs of the cells being cultured.
Cytokines and growth factors are involved in a number of critical cellular processes including proliferation, adhesion, morphologic appearance, differentiation, migration, inflammatory responses, angiogenesis, and death. A list of soluble factors currently used for cell cultivation includes, but is not limited to, the following growth factors and cytokines: EGF, HGF, IGFII, TGFbeta, FGF, VEGF, PDGF, IL-3, IL-6, G-CSF, GM-CSF, SCF, IL-15, IL-11, NGF, erythropoietin etc.).
Alternatively, in order to favour the cultivation of precursor cells, and in particular for those of hematopoietic origin, stromal cells lines are used as feeder cells (Kittler E L et al. Blood. 1992; 22:3168-3178. Eaves C J. et al. Blood. 1991; 78:110-117. Krebsbach P H. et al. Crit Rev Oral Biol Med. 1999; 10:165-181; Charbord P. et al. Exp Hematol. 2002; 30:1202-1210).
The above mentioned methods of cultivation have several drawbacks.
Culture media supplementation with specific soluble factor cocktails, that appears an absolute requirement for the maintenance and/or differentiation towards specific differentiate programs of stem cells is expensive.
The major drawback of cultures utilizing feeder cells is the difficulty of procedures for the separation of the generated cells from the feeder cell. Indeed, the generated cells may be partially adherent to the feeder cells and the manual separation of the two cellular types results in an extremely hard-work. Moreover, the yield of recovered cells is often affected.
In order to solve these problems, the use of some media conditioned by different cell types has been proposed. When incubated with specific cell types, the culture medium becomes to those skilled in the art as “conditioned medium”. Conditioned media contain many of the original components of the medium, as well as a variety of cellular metabolites and secreted proteins including, for example, biologically active growth factors, inflammatory mediators and other extracellular proteins specifically released by the “conditioning cells”. The exact composition and therefore biological properties of each conditioned medium strictly depends on the conditioning cells.
The present invention relates to the preparation and use of a medium/conditioned by MMH cells.
The MMH cells have been described for their phenotypic and functional characteristics in several publications (Amicone L. et al EMBO J. 1997; 16:495-503. Spagnoli F M. et al. J Cell Biol. 1998; 143:1101-1112. Bellovino D. et al. J Cell Physiol. 1999; 181:24-32. Spagnoli F M. et al. J Cell Sci. 2000; 113:3639-3647. Napolitano M. et al. Free Radic Biol Med. 2001; 30:506-515. Pasquetto V. et al. J Virol. 2002; 76:5646-5653.).
It is known from Aiuti A. et al., Hepatology. 28:1645-1654 (1998) that MMH cells may be used in a cell-cell contact co-culture system with liver cells derived from 15 days post-coitum mouse embryos. It has been shown that MMH cells are able to sustain maintenance, proliferation and differentiation of hematopoietic embryonic cells; the direct contact between hepatocytes and hematopoietic cells constitutes therefore a specific culture requirement in that the cells need to be made to grow in contact with the MMH cells. Yet the co-culture has drawbacks in the analysis and use of cells so generated since this procedure requires, after the growing step, a further separation step from MMH cells. Since the hematopoietic cells partially adhere to MMH cells, the separation of the two cellular types results, as described above, in an extremely hard-work, and an inefficient number of the hematopoietic cells recovered. Moreover, the use of embryonic cells highly limits the realization of the method because of bioethics problems and difficulties in material availability.
It is known from Penington G D (Blood Cells. 1979. 5:13-23) that it is possible to cultivate bone marrow cells in a medium conditioned by hepatocytes. It is shown that the rat liver cell line BRL-3A is able to sustain the growth of megakaryocyte colonies. However this method has several drawbacks, in that BRL-3 cells have been demonstrated not to be fully differentiated hepatocytes, as revealed by the morphology of the cells and, mainly, by the lack of expression of many liver-specific genes, fundamental to retain the complex hepatic functions in vitro (Boess F et al. Toxicol Sci. 2003. 73:386-402). In addition, BRL-3A-conditioned medium has been shown to induce transformation of some mammalian cells (Massague J et al. J Biol. Chem. 1985. 260:4551-4554).
In order to improve the quality of hepatocytic conditioned media other researcher used different sources of conditioning cells. Hepatoma cells have been used (Nakamura H et al. J Biol. Chem. 1994. 269:25143-25149) but they are transformed cells. Moreover, these cells are not able to sustain the survival and/or the differentiation of mammalian cells. Allen K J et al. (J Gastroenterol Hepatol. 2000. 15:1325-32) generated conditionally immortalized mouse hepatocytes using a simian virus 40 (SV40) large T antigen (TAg) gene. SV40 in humans is associated with inflammatory kidney diseases and with specific tumor types: mesothelioma, lymphoma, brain, and bone. These human tumors correspond to the neoplasms that are induced by SV40 experimental inoculation in rodents and by generation of transgenic mice with the SV40 early region gene directed by its own early promoter-enhancer (for a recent review see Barbanti-Brodano G. Virology. 2004. 318:1-9). So these immortilised hepatocyte are potentially oncogenic transformant and for these reasons they are not suitable for the culture of cells. Other media conditioned by primary hepatocytes isolated from adult rats are described in Krause P et al. (J Hepatol. 2000. 32:718-726) Haupt W et al. (Life Sci. 2000. 67:3191-3198). However such media have several drawbacks: i) primary hepatocytes survive only few days in culture; ii) normal hepatic gene expression is downregulated in primary hepatocytes iii) in order to produce the hepatocytes-conditioned medium at industrial scale too many animals are required to be sacrificed; iv) the isolation of primary hepatocytes from the liver is an hard work and is time and costs consuming.